In the highly regulated production of pharmaceuticals, a large expenditure in terms of time, equipment and personnel is apportioned to the provision of cleaned and sterilized bioreactors. In order to avoid cross-contamination reliably in a product change in a multipurpose plant or between two product batches, apart from the cleaning, a very complex cleaning validation is required which may need to be repeated in the event of a process adaptation. This applies not only to upstream processing, USP, that is to say the production of biological products in fermenters, but also to downstream processing, DSP, that is to say purification of the fermentation products. In USP and DSP, use is frequently made of kettles as agitator and reaction systems. Especially in the case of fermentation, an aseptic environment is essential for successful culturing. For the sterilization of batch or fed-batch fermenters, generally the SEP technique is used. In order in the case of continuous process procedure to ensure sufficient long-term sterility, the autoclave technique is also used, which however, requires laborious transport of the reactors to the autoclave and is only usable with comparatively small reactor scales. The risk of contamination during fermentation is particularly critical during sampling and at moving stirrer shafts. The latter are generally equipped with complex sealing systems (e.g.: sliding-ring seals). Technologies which succeed without such penetration of the fermentation casing are preferred because of their greater process robustness.
The downtime of the reactors necessitated by the preparation procedures can be, in particular in the case of short use periods and frequent change of product, of the order of magnitude of reactor availability. The affected steps in the USP of biotechnological production are the process steps of media production and fermentation, and in the DSP, solubilization, freezing, thawing, pH adjustment, precipitation, crystallization, buffer exchange and virus inactivation.
For carrying out the reactions in the USP and DSP, frequently a plurality of reaction conditions must be met simultaneously. For instance, fermentation, for example, in addition to oxygen supply and CO2 removal, requires gentle suspension of the cells, rapid mixing of the media and neutralizing agents for avoidance of overconcentration and also heating of the reaction liquid. Particle retention can also be required, e.g. for the use of perfusion strategies.
In the case of precipitation and crystallization, rapid addition of the precipitant, efficient temperature control and gentle holding of the particles formed in suspension are of particular importance.
Generally, in all process steps of biotechnological production, shallow temperature gradients are required in order not to damage the products. This condition, especially in freezing and thawing processes, leads to considerably increased process times with increasing reactor scale, since no mixing elements can be used in these steps. Heat transport into the reaction medium is limited by the thermal conductivity of the ice layer and also by free convection in the liquid. Long process times, however, can lead to considerable product losses in the presence of proteolytic activity.
Gentle sterilization and virus inactivation of starting materials and product solutions can be achieved by UVC irradiation at a wavelength of 254 nm. The radiation damages the DNA and RNA which lie at the absorption maximum of the viruses and microorganisms and prevents thereby their multiplication, whereas the proteins which are situated in the absorption minimum of the UVC radiation are very substantially retained. A great problem is the depth of penetration of the UVC radiation, which is frequently restricted to only a few tenths of a millimeter in biological media. This makes efficient replacement of the film in the active irradiation zone necessary in order firstly to irradiate all viruses with the required radiation dose and secondly to minimize the radiation load of the products.
The requirement of a constantly renewed boundary layer is also posed in the case of filtration, in order to counteract the development of covering layers which limit the transmembrane flow.
All process engineering steps of mass transport and heat transport, particle separation, UV irradiation and the addition or distribution of solids or additives or gases requires sufficient agitation of the reaction medium. This agitation is ensured, in the pharmaceutical industry, in the stainless steel reactors customarily used there, by means of appropriately dimensioned agitators or by sparging.
Membrane gas introduction is used for gentle oxygen supply of cell cultures. As membranes, gas-permeable silicone tubes are wound onto a cylindrical membrane stator which receive flow from a radially-transporting anchor agitator [WO 2005/111192 A1]. A more than doubling of the exchange area and thereby a significant increase in mass transport can be achieved by paralleling the membrane stators.
Other membrane gas-introduction systems [WO 85/02195 and DE 10 2004 029 709 B4 and DE3428758], in the gas introduction, set up agitators or baskets which are covered with membrane tubes and are moved in a pendulum-like manner in the fermentation solution, or membrane stacks [U.S. Pat. No. 6,708,957 B2], which are swung in the fermentation solution. These membrane gas-introduction systems, however, are distinguished in that they can only be converted to an industrially relevant scale with limitations.
In order to meet the demand for a rapid and flexible charging of the production plant while maintaining maximum cleanliness and sterility, designs for single-use reactors are the subject of constantly growing interest on the market.
Single-use technologies for filtration have long been known. Recently, a single-use technology has also become available on the market for UVC treatment [WO02/038191 WO02/0385502, EP1464342]. Designs for single-use heat exchangers are only available for small scales [EP1464342]. All technologies are operated in continuous flow, so that in addition to a reservoir vessel, the use of pumps and lines is necessary for which cleaning and sanitation plans still need to be provided as before.
There currently exist various commercially available mixing systems which operate on the basis of a plastic bag—single-use technology. These include systems [Hyclone Laboratories, Inc. (http://www.hyclone.com)] which are equipped with blade or magnetic stirrers or circulation pump elements. The systems are available up to a volume of 200 L. [Sartorius AG (http://www.sartorius.de)] offers a single-use system which operates up to a volume of 500 L using a free-floating single-use magnetic stirrer which has no contact with the single-use plastic bag and therefore also no material wear. Single-use mixing systems up to a volume of 10 liters are available at [ATMI, Inc (http://www.atmi-lifesciences.com)]. In this system the material to be mixed is charged into a single-use bag and mixed under rotation. For larger volumes up to 200 L [ATMI, Inc.] offers a single-use bag stirring system which is distinguished in that the stirring element is invaginated into the bag. Mixing in this case is not achieved by a rotary motion around a fixed axis but by a stirring-tilting motion.
In [EP 1 462 155 A1], use is made of a single-use vessel for mixing and dispersing materials by means of a magnetic stirrer which is situated within a protective cage in order to prevent damage to the plastic bag. The product-contact region of the magnetic stirrer unit in this case likewise consists of single-use components.
[EP 1 512 458 A1] demonstrates a solution in which inflatable plastic pillows are integrated in the external or internal region of a single-use bag system. These pillows are alternately pressurized and depressurized again. This induces liquid movements which lead to intensification of mixing and suspension in the vessel.
There are a multiplicity of patents for the use of single-use technology in the fermentation technique sector. In these, in most systems mixing and oxygen supply are achieved via sparging, without further mixing systems being provided [U.S. Pat. No. 5,565,015, WO 98/13469, U.S. Pat. No. 6,432,698 B1, WO 2005/049785 A1, EP 1 602 715 A2, WO 2005/080544 A2]. If a higher oxygen demand is necessary for the culture which cannot be achieved alone via sparging, the sparging can be combined with a dispersing mixing system [WO 2005/104706 A2, WO 2005/108546 A2, WO 2005/118771 A2] or can be overlapped by pumped circulation [WO 2005/067498 A2]. The maximum process volume of a sparged unit is currently up to 1000 liters. In systems having conventional agitators, but which can also be designed as single-use systems [WO 2005/104706 A2, WO 2005/108546 A2], process volumes of up to 10 000 L are achieved.
In the case of sparging, foaming problems can make the use, and the subsequent complex removal, of antifoams necessary in the DSP. The cell stress on bubble rise, in the bursting of the gas bubbles at the surface, and in particular in the foamed destruction, is problematic in cell culture systems, since the cells can be permanently damaged by the resultant high shear forces which are introduced. This applies all the more when sparging is combined with a dispersing agitating system, that is to say an agitating system comminuting the gas bubbles. The damaged cells release proteins, the removal of which can lead to considerable product losses during workup. To maintain acceptable cell vitalities, the oxygen input into the abovementioned bioreactors and therefore also the cell density which is achievable must be restricted. The restricted cell density ultimately reduces the space-time yield of the fermenters and the capacity of the total plant. Since a precondition for reliable upscaling in most cases is not considered technically as met, in the sparged single-use reactors, the volume enlargement must be achieved by complex paralleling of the systems. If the fermenters are operated as proposed using standard agitating systems, although the volume which can be processed increases into the range of the permanently installed plants, the risk of contamination can only be managed with comparable technical expenditure, for example by the use of damped sliding-ring seals. The great technical complexity and expenditure on personnel of such installations, however, largely emphasizes the advantages of the single-use concept.
Other single-use systems provide the necessary gas-introduction rate of the culture by means of membrane or surface gas introduction. In this case the necessary exchange area for gas transport is provided either via a membrane permeable to the gases to be transferred, or via an open boundary area to a gas space. Since no direct gas introduction to the cell culture media proceeds, the particle stress in these reactors may be categorized as low.
[U.S. Pat. No. 5,057,429] describes a system in which an inner semipermeable flat bag which is filled with cell suspension is surrounded by a further bag which is filled with nutrient solution and enriched with oxygen. Nutrient transport and oxygen transport are intensified via a tilting motion of the bags. The maximum process volume of a unit is only a few liters. The oxygen input is considerably restricted by the low oxygen solubility in the charged medium and the comparatively low surface area of the membrane. Compared with standard membrane gas-introduction devices [WO 2005/111192 A1] having specific exchange areas in the order of magnitude of 30 m2/m3 in 100 L reactors, in this arrangement, only a maximum of 10% of this exchange area can be achieved. In both cases, the available exchange area, furthermore, decreases in proportion to the scale enlargement.
Other surface gas-introduction systems likewise operate with a flat bag which is clamped on a shaking apparatus. The bag is only partially filled so that a free surface having a gas space lying thereabove is formed. By means of a seesawing motion or an eccentric rotary motion, the culture medium is mixed, the nutrients which are fed are distributed, cell sedimentation is prevented and the surface is agitated [U.S. Pat. No. 6,190,913 B1, WO 00/66706, U.S. Pat. No. 6,544,788 B2]. In this technology the culture is supplied with oxygen via the free surface. The motion is continuously adapted in such a manner that the flow is gentle and the cells are not exposed to strong shear. The maximum process volume of a unit is currently 580 liters. Although this technology provides a gentle gas-introduction mechanism, it is restricted in conversion to an industrial scale. The height of the bag must be kept approximately constant so that an increase in volume at constant surface area to volume ratio can only be achieved in the two horizontal spatial directions. Upscaling can therefore only be achieved via technically complex paralleling.
The technologies available on the market make use, for freezing, of large stainless steel reactors which are supplied with cooling liquids, or small flat plastic bags which are frozen in a secondary manner via heat-conducting surfaces or by means of convective cold air. In both cases there is no possibility of agitating the product during the freezing process, which considerably lengthens the cooling and freezing processes. The metal vessels are expensive and require large storage areas in the temporary storage. Thawing is lengthy, since the liquid motion between ice block and container wall proceeds only via free convection, comparably to that in freezing. For thawing the plastic bags, these are cut open in the frozen state and subsequently charged into a stirred reactor. The procedure of cutting them open is labor-consuming and contributes to fouling of the working environment. The thawing process is time-intensive, because the ice blocks which float on the surface are hardly reached by the hydrodynamics prevailing in the reactor. Product losses in the course of the long thawing phases are therefore unavoidable.
In the employment of all of the reactors listed here, considerable losses must be accepted in performance and upscalability. In many cases, without sufficient scalability, apart from the lack of performance, an economic benefit cannot be guaranteed. Scaleup here can only be achieved at the cost of increasing complexity and decreasing the economic benefit, such as, for example, by paralleling a plurality of reactors or by the additional use of technically complex solutions (for example sliding-ring seals built into the plastic bags).
A reactor which can be scaled up to the industrial scale of 1 m3-10 m3, guarantees a very high level of sterility comparable to autoclaving by avoiding shaft seals and the problems of cleaning, permits simultaneously intense and gentle liquid motion and can be installed with low expenditure on equipment and personnel, is therefore a clear gap in the currently available range of technologies.
It was an object of the present invention to produce a reactor, in particular for pharmaceutical applications, which, even on large reactor scales, has very good reaction properties for carrying out biological, biochemical and/or chemical reactions with respect to mixing, distribution, suspension, solubilization, mass transport and heat transport, filtration and irradiation, or combinations thereof, and is preferably simple in handling, meets the high requirements of the pharmaceutical industry with respect to cleaning and sterility and contributes to increasing process robustness and to increasing the space-time yield.